WO1984002848A1

WO1984002848A1 - Anti-idiotypic antibodies to t cell antigen receptors

Info

Publication number: WO1984002848A1
Application number: PCT/US1984/000111
Authority: WO
Inventors: Nicole Suciu-Foca; Patrick C Kung
Original assignee: Centocor Inc; Univ Columbia
Priority date: 1983-01-28
Filing date: 1984-01-27
Publication date: 1984-08-02
Also published as: EP0135536A1

Abstract

Anti-idiotypic antibodies against antigen receptors (idiotypes) on the surface of human T lymphocytes, methods for producing these anti-idiotypic antibodies and diagnostic and therapeutic uses for them. The anti-idiotypic antibodies of the invention react with human T lymphoblasts which have been activated by alloantigens in vivo or in vitro. It has been discovered that alloactivation in vivo through pregnancy induces the formation of antiidiotypic antibodies in mothers which are specific for receptors (idiotypes) on the surface of T cells. The anti-idiotypic antibodies against a T cell surface receptor inhibit the specific reactivity of antibodies produced by B-lymphocytes against the antigen recognized by the surface receptor. For example, anti-idiotypic antiserum from parous mothers inhibits the cytotoxic activity of alloantisera toward father's lymphocytes. (Fig. 1 and 2) Auto-anti-idiotypic antibodies also inhibit the autologous mixed lymphocyte response to the mother's own lymphoblasts alloactivated by the hather's lymphocytes, a phenomenon whose in vivo correlate may be the auto-inhibition of anti-HLA antibody formation and of allograft immunity.

Description

ANTI-IDIOTYPIC ANTIBODIES TO T CELL ANTIGEN RECEPTORS

Description

Technical Field This invention is in the field of cellular immunology and relates to antibodies to antigen receptors (idiotypes) on the surface of human T lymphocytes.

Background of the Invention There are three categories of antigenic determinants which are used to classify antibodies. Those that differentiate between the main immuno- globin classes are the same in all individuals of a species and are called isotypes. Another class of determinants reflect small differences between individuals of the same species in the amino acid sequences of similar immunoglobulins and are called allotypes. Allotypes are detected with homologous antiserum or alloantisera, which are formed by one individual against immunoglobulins with different allotypes from another individual of the same species.

The third kind of antigenic determinant is an idiotype. These are determinants which are unique to the immunoglobulin molecules produced by a given clone of immunoglobulin producing cells. Idiotypes appear to be located on the binding site of the . immunoglobulin. They may be detected with antisera produced by one individual against an antibody from another individual of the same species and with the same allotype.

OMPI - The products of HLA system, the major histo- compatibility complex in man, are alloantigens which like immunoglobulin allotypes differ between indi¬ viduals of the same species. The HLA system pro- duces two types of products. The products of the HLA-A, -B and -C loci, the class I genes, are ubiquitous; they are present on almost all cells of an organism, and they elicit rejection of trans¬ planted tissue by the immune system of the reci- pient. In contrast, the products of the D/DR locus are present only on certain types of immune cells and are involved in regulating antibody production by B-lymphocytes.

Human subjects alloim unized through pregnancy, blood transfusion or organ transplantation develop antibodies directed against the gene products of HLA. Surprisingly, however, only about a quarter of multiparous women have anti-HLA antibodies even though all such women have been exposed to the paternal HLA alloantigens of the fetus which cross the placental barrier during pregnancy. Further, kidneys which are transplanted from children to their mothers or fathers survive about 75% of the time, a finding which indicates that most mothers are not immunized to their children. Opely, G. and Terasaki, P.I., Transplantation 24;106 (1977).

Even more surprising is the finding that only 30% of dialysis patients who have been transfused repeatedly with blood from a single donor, develop anti-HLA antibodies. Kidneys transplanted from the actual blood donor into a recipient who did not form anti-HLA antibodies in response to the repeated blood transfusions, show the highest rate of

OMPI survival. Salvatierra, O. ei: al. , Transplantation 24 : 106 (1977) .

Observations such as the aforementioned have generated the concept that human subjects may be categorized with respect to their iramunogenic response to alloantigens as either "high responders" or "low- or non-responders". The outcome of a tissue transplant, it is thought, depends upon the particular state of responsiveness of the recipient. A "high responder" recipient would have a greater tendency to reject transplanted tissue than would a "low- or non-responder" recipient. The factors that determine responsiveness or non-responsiveness to transplantation antigens are not clearly understood. A method of inducing a state of non-responsiveness in a recipient of a transplant would greatly in-, crease the likelihood of survival of the trans¬ planted organ. One of the objects of this invention is a method of suppressing allograft immunity by inducing a state of nonresponsiveness in prospective recipients of a transplanted organ. In order to fully understand the present invention and how such suppression of allograft immunity may be brought about, the role of lymphocytes in the mediation of immunity must be examined.

All lymphocytes can be split functionally into two main groups, B lymphocytes and T lymphocytes. B lymphocytes differentiate in the Bursa of Fabricius (in birds) or possibly in the bone marrow (in mammals) and become antibody secreting plasma cells. T-cells mature under the influence of the thymus gland. Upon encountering an antigen, B lymphocytes differentiate into plasma cells which secrete antibodies against the antigen; this is known as

OMPI antibody-mediated immunity. T cells however exert their effects directly; this is cell-mediated immunity.

There are three important subpopulations of T cells: helper cells, which interact with B cells to amplify production of antibody, effector cells, which carry out the direct cell-killing function of T cells and make certain lymphokines (non-antibody products) which are responsible for delayed hyper- sensitivity, and suppressor cells, which participate in the regulation of both antibody-mediated and cell-mediated immunity. T cells must be activated before any of these forms of activity are expressed. Usually the activation follows from exposure to antigen, but other less specific factors such as interleukin 2 are also believed to participate in the activation of T cells.

Usually there is a latent period of around a week to 10 days after first exposure to antigen before the T cells develop initial reactivity.

Shortly after thatv the reactivity subsides. But upon a second contact with antigen, T cells show an accelerated memory response with high activity developing within 2-5 days. An important feature of a mature lymphocyte is its unique surface receptor which enables it to recognize a particular antigen, actually a parti¬ cular antigenic determinant. An individual lymphocyte responds to only one antigenic deter- minant because it has only one type of a surface receptor. This special correspondence means that the immune response to an antigen is highly specific. Both B and T cells manifest their response to antigens by developing clones. When a T cell, for example, encounters the antigen which its receptor recognizes, it enlarges into a lymphoblast. This lymphoblast then goes on ^'to divide sequentially and exponentially giving rise to a clone of T lympho¬ cytes with identical specificity. This process is known as clonal selection and expansion.

The addition of antigen to cultured lymphocytes induces a small proportion of T-cells to differ¬ entiate into the larqe rapidly dividing blast cells. T lymphocytes can also be transformed by culturing them together with the lymphocytes of individuals of the same species, a so-called mixed lymphocyte culture (MLC) . Because of extensive polymorphism at HLA loci, the two cell populations are virtually always different antigenically and they stimulate each other to undergo blast transformation. For example, isolated blood lymphocytes from recipient and prospective donor are maintained together for several days in^' issue culture. Blast transforma¬ tion occurs if allogeneic cells are present. Such . blasts may be referred to as alloactivated lympho¬ blasts. One-way LC's may be established by treat- ing one set^"of cells in a manner that prevents blast transformation of that set of cells such as irradiating the cells.

A T lymphocyte will recognize an antigen only if the antigen is properly presented by a presenting cell which in many cases is a macrophage. The antigen must be presented juxtaposed to a compatible la molecule, a surface molecule coded for by one of the class I transplantation or histocompatibility genes. In man, there are at least two distinct families of la molecules, HLA-DR and MT or DS. These genes control the formation of the specialized complementary la structures on the surface of a presenting cell and the T cells that provide for proper presentation of antigens. T cells may interact with B cells, or other T cells, if the cell possesses complementary la structures and if it recognizes the same antigenic determinant or a different determinant on the same antigenic ole- cule.

Idiotypes on the antigen binding receptors on both B-cells and T cells have been shown to function in their own right as antigenic stimuli which induce the formation of anti-idiotypic antibodies. The reaction between idiotypic receptors and anti- idiotypic receptors is thought to be a major factor in regulating a specific immune response. See e.g. Wigzell, H. and Bing, H. , In: Progress in Immunology IV, eds. Fougereau, . and Dausset, J. (Academic Press, N.Y.) p.94-103 (1980); Infante et al. J. Exp. ed. 155:1100 (1982); Bona, C. and Paul, E. , In: Regulatory T Lymphocytes, eds., Pernis, B. and Vogel, H.J. (Academic Press, N.Y.) p.292 (1980). This invention relates to anti-idiotypic antibodies directed against antigen receptors on the surface of human T lymphocytes.

The receptors on the surface of T cells have several domains. The domain which is unique to each type of receptor is called the idiotype; it is located in the variable region of the receptor and is analogous to the idiotype region of antibodies. Other domains of the receptor are shared by all types of receptors. Hereinafter the term idiotype is used interchangeably with the term receptor

OMPI because for purposes of the invention, the idiotype domain on the receptor is the most important region, Disclosure of the Invention

This invention relates to anti-idiotypic antibodies against antigen receptors (idiotypes) on the surface of human T lymphocytes, methods for producing these anti-idiotypic antibodies and diagnostic and therapeutic uses for them.

The anti-idiotypic antibodies of the invention react with human T lymphoblasts which have been activated by alloantigens _in vivo or _in vitro. It has been discovered that alloactivation _in vivo through pregnancy induces the formation of auto- antiidiotypic antibodies which are specific for receptors (idiotypes) on the surface of T cells. The anti-idiotypic antibodies react with T cell receptors for alloantigens and are able to inhibit the reactivity of antibodies against alloantigen recognized by the idiotypic T cell receptor. Brief Description of the Drawings

Figure 1 shows the inhibition of the lympho- cytotoxicity of anti-ALA-DR2 alloantiserum 8W907 by auto-anti-idiotypic antibodies.

Figure 2 shows the inhibition of anti-HLA antibody by serum containing auto-anti-idiotypic antibodies. An early (4 month) post delivery serum from a biparous woman (ARF) who developed anti-HLA- -B7 and anti-HLA-B22 antibodies reacted with 95% T lymphocytes from her husband at up to a 1:64 dilu- tion (O—o). The binding was significantly inhi¬ bited when the serum was preincubated with a late post-delivery (4 years) serum in which the anti-HLA antibodies reacting with the husband's lymphocytes were no longer detectable (•—•) .

_OMPI Best Mode of Carrying Out the Invention

Sera from parous women contain auto- anti-idiotypic antibodies which react with auto¬ logous alloactivated lymphoblasts which have been activated in vivo against the f ther*s HLA allo¬ antigens, that is, the sera react with lymphoblasts expressing receptors for the father's alloantigens.

Further the anti-idiotypic antibodies against a T cell surface receptor inhibit the specific reac- tivity of antibodies produced by B-lymphocytes against the antigen recognized by the surface receptor. Thus, the serum from a mother which binds to autologous alloactivated lymphoblasts is also capable of inhibiting the anti-HLA activity of alloantisera specific for the father's allotype inherited by the child. Also, auto-anti-idiotypic antibodies inhibit the autologous mixed lymphocyte response to the mother's own lymphoblasts alloacti¬ vated by the father*s lymphocytes, a phenomenon whose jLn vivo correlate may be the autoinhibition of anti-HLA antibody formation and of allograft immu¬ nity. It may explain why a mother is not immunized against her child.

The anti-idiotypic antibodies of this invention may be used in assays to detect specific antigen receptors on the surface of human T lymphocytes. These assays would be based upon conventional methods of detecting the presence of antigens on the surface of cells, such as radioimmunoassay, enzy e- linked-immunoadsorbent assay, or immunofluorescent assay. Where necessary, the anti-idiotypic anti¬ bodies may be labelled with a radioisotope, an enzyme, fluorescent compound, etc. In addition, such assays may be used to detect T cell surface receptor molecules which have been secreted or shed by the T cell and are present in blood, lymph or other biological fluid. Well-known methods for detecting soluble antigens would be used.

The antibodies of this invention may also be used to isolate from a mixed population of human T lymphocytes those T lymphocytes bearing specific receptors upon their surface. For example, an antibody affinity column or other type of immuno- adsorbent device could be constructed using anti- idiotypic antibodies against specific antigen receptors on the surface of T cells. Such immuno- adsorbents may selectively adsorb cells having the receptors.

Finding that alloimmunization leads to the development of auto-antibodies against T cell receptors for allogeneic HLA-DR antigens, suggests that the receptor for self and for non-self la must be different. If the receptors were the same, then auto-anti-idiotypic antibodies developing following alloimmunization would block the T cell recognition system. This would imply that pregnant women, for example, fail to recognize T cell dependent anti¬ gens, i.e., that alloimmunization leads to T cell deficiency. This is not the case. Sera from women who have been alloimmunized through pregnancy against the same haplotype are capable of binding to autologous as well as to each other's lymphoblasts, i.e., their receptor for the immunizing allogeneic DR antigen and their anti- receptor antibodies are cross reacting. Also, a serum tested for the presence of anti-idiotypic antibodies against anti-HLA-DR2 antibodies, and found to react with AL primed against DR2, also inhibited the lymphocytotoxic activity of two anti-HLA-DR2 alloantisera. The anti-anti-HLA receptor expressed by T and B lymphocytes seem therefore to share common determinants and to display little variability from one individual to antoher, a finding with great clinical implications as far as the potential usefulness of anti-anti-HLA antibodies for iramunotherapy is concerned.

Anti-idiotypic antibodies were found to be present both in women who have developed anti-HLA antibodies and in those who did not. The capacity of anti-idiotypic antibodies to inhibit the anti-HLA antibody activity of earlier sera from the same donor, was documented in one case. This suggests that "responsiveness" versus "non-responsiveness" to transplantation antigens, reflects the balance between anti-HLA and anti-anti-HLS antibodies. When the former dominate, the presence of the latter will be simply overseen, while when the anti-anti-HLA antibodies prevail, the balance will swing to what appears to be "unresponsiveness" to alloantigens. This phenomeno could well account for the absence of HLA antibodies in allosensitized individuals.

An extremely significant therapeutic use for the anti-idiotypic antibodies is in the suppression of allograft immunity. By passive transfer of antireceptor immunity by way of anti-idiotypic antibodies, allograft immunity may be specifically suppressed. For example, a prospective recipient could be injected with anti-idiotypic antibodies of defined specificity so that cells bearing receptors (idiotypes) to certain alloantigens could be killed and the immune response to those alloantigens abolished. For example anti-anti HLA antibodies of defined specificity could be obtained from

-£fREΛ OMPI multiparous women and used to eliminate lymphocytes of that specificity.

In addition these antibodies may also be used in the treatment of various autoimmune diseases. The antibodies could be used to selectively elimi¬ nate those T lymphocytes which are responsible for mediating the immune reaction against self.

As indicated above, the anti-idiotypic anti¬ bodies of the present invention may be obtained from the sera of alloimmunized individuals such as parous women or the recipients of blood transfusion. However as would be evident to one skilled in the art, monoclonal antibodies can be produced employing cells which produce antibodies to T cell antigen receptors. Basically these techniques involve the fusing of the antibody producing cell with an "immortal" cell, such as a myeloma, to provide a fused cell hybrid which has immortality and is capable of producing the desired antibody, in this case an antibody against a specific idiotype on the surface of T lymphocytes. The hybrid cells are cultured under conditions conducive to the pro¬ duction of antibody which is subsequently isolated from the cell culture medium. Such techniques are well described in the literature. See, for example, U.S. Patent No. 4,196,265 issued to Hillary Koprowski e_t al. , the teachings of which are hereby incorporated by reference.

Exemplification

Donors

Heparinized and nonheparinized blood was obtained from healthy parous women, from their husbands and from unrelated controls. All indivi¬ duals were typed for HLA-A, B, C and DR using the lymphocytotoxicity method and a selected battery of anti-HLA alloantisera.

Mixed Lymphocyte Cultures

Lymphocyte suspensions were prepared by the

Ficoll Hypaque gradient centrifugation method.

7 One-way MLC reactions were set-up using 1 x 10

7 responding lymphocytes and 1 x 10 irradiated (200Or) stimulating lymphocytes in 20ml RPMI 1640 medium supplemented with glutamine, antibiotics and 25% pooled normal male serum. Alloactivated lymphoblasts (AL) were obtained from 5-day-MLC cultures by differential centrifugation through 55% and 30% isotonic Percoll suspensions.

Cytofluorometric determinations of antibody binding to MLC alloactivated lymphocytes

Lymphocytes from 5-day-MLC were suspended in RPMI 1640 medium supplemented with 30% horse serum (HS) and incubated for 18 hours at 37°C in a humidified 5% C0₂ atmosphere. Cells were then washed twice and adjusted to a concentration of 5 x 10 lymphocytes/0.1 ml of medium to which 0.1 ml of test serum was added. Following 30 minutes of incubation at 25°C and 30 minutes at 4°C the

++ +-- suspens on was washed three times in cold Ca Mg free phosphate buffered saline (PBS) containing 5% fetal calf serum and 0.05% sodium azide (PBS-FCS buffer) . The cells were then incubated for 30 minutes at 4°C in 0.1 ml of a F(ab₂') sheep-anti- human immunoglobulin xenoantiserum conjugated to FITC (Central Laboratory of the Netherlands Red

OMPI Cross Blood Transfusion Service) supplemented with

10% horse serum. Cells were washed and subjected to flow cy ometrie analysis using the ORTHO SPECTRUM

III. Binding of autologous serum to AL was ex- pressed as percent binding of autologous serum minus percent binding of pooled normal male sera. The latter ranged from 0 to 30% for different samples.

Indirect rosetting assay

Lymphoblasts obtained from 5-day-MLC were washed twice and resuspended at a concentration of 1 7 x 10 cells/ml in RPMI 1640 medium. An equal volume of test serum was added and the suspension was incubated for 1 hour at 4°C. Cells were then washed

3 times and resuspended in 1 ml of RPMI 1640 to which 1 ml of a 0.5% suspension of sheep erythro- cytes (SRBC) pretreated with purified goat-anti- human IgG and chromium chloride was added. The mixture was incubated for 45 minutes at 4°C and for 15 minutes at 37°C, and the percentage of rosetted and non-rosetted viable lymphoblasts was determined under phase-contrast microscopy. Determination of MLC blocking activity of the sera

Sera were tested for their capacity to suppress the allogenic MLC reaction displayed by the mother's lymphocytes against irradiated, "stimulating" lymphocytes from her husband and from selected controls. In parallel, sera were tested for their capacity to inhibit the autologous MLC response (AMLR) displayed by T lymphocytes against autologous T lymphoblasts, that have been alloactivated in a

5-day MLC against the immunizing donor (husband) or against unrelated controls. Triplicate reactions containing 5 x 10 4 responding cells and 5 x 104 irradiated stimulating cells were grown for 5 days in the presence of 25% test serum, or normal male serum. The amount of tritiated thymidine incorpor¬ ated was quantitated on day-6 and the percent MLC inhibition induced by the test serum, compared to the normal male serum was calculated.

Determination of lymphocytoxic anti-HLA antibodies in sera from parous women. Nylon-wool-column-purified T and B cell suspen¬ sions from the immunizing donor and from an HLA reference panel were used as targets for ascertain¬ ing the presence of lymphocytotoxins reacting with Class I (HLA-A, B, C) or with Class II (HLA-DR) histocompatibility antigens. The cytofluoroch.ro- masia technique for complement-dependent lymphocyto- toxicity testing was used.

Inhibition of anti-HLA lymphocytotoxic activity of alloimmune sera by anti-idiotypic antibodies. Sera from 3 women who showed no anti-HLA antibodies and sera from 5 women with antibodies against Class I and/or Class II antigens were tested for their capacity to react with AL which had been alloactivated in MLC against the immunizing donor (husband) or against unrelated controls (Table 1) . Binding of the serum to AL alloactivated against the husband occurred in all cases. In no instance was binding observed when the serum was tested on resting autologous T lymphocytes. TABLE 1 . REACTIVITY OF ALLOACTIVATED T-LYMPHOBLASTS (AL) WITH AUTOLOGOUS SERUM

INFORMATION ON MATERNAL SERUM MLC COMBINATION % BLASTS NO .

CASE (HLA-DR , MT-HAPLOTYPES) IN DAY 5 PREGNANCY

RESPONDER STIMULATOR MLC

* *

M DR7 MT3 F . DR2 MT1 3

DR7 MT3 DR6 MT1 MT2

C⁺ DR1 MT1 3 DR3 MT2

2 M DR2MT1 F DR6MT1MT2 3 2

DR5MT2 MT4 DR7MT3 3 M DR5MT2 M 4 F DR6MT1MT2 5 2

DR7MT3 DR2MT1

4 M DR7M 3 F DR6MT1MT2 22 3

DR7MT3 DR2MT1

C DR6MT1MT2 10 DR5MT2 MT4

C DR7MT3 13 DRX

M DR6MT1MT2 F DR2MT1 8

DRX DR6MT1MT2

C DR5MT2 MT4 5 DR6MT1MT2

C DR7MT3 17 DRX

M DR4MT3MT4 F DR4MT3MT4 3

DR7MT3 DR5MT2MT4

C DR4MT3MT4 6 DR3MT2

M DR1MT1 F DR3MT2 3

DR3MT2 DR4MT3MT4

C DR5MT2MT4 DR8MT2

M DR4MT3MT4 F DR1MT1 7

DR6MT1MT2 DR6MT1MT2

^• C DR7MT3 3 DR7MT3

M * * Mother

Father

Control -ISA-

Table 1 (Cont.) REACTIVITY OF ALLOACTIVATED

T-LYMPHOBLASTS (AL) WITH AUTOLOGOUS SERUM

INFORMATION ON MATERNAL SERUM TIME POST ANTI- % A.L. REACTING WITH

CASE LAST DELIVERY HLA ANTIBODIES AUTOLOGOUS SERUM

DR ABC MOTHER FATHER

-4 MO. none none 45

24

2 32 YRS. none none 11 0

3 7 MO. none B7, B22 28 0

4 2 YRS. MT2 none 26 0

1

2 MO. none A3 ,B7 14

0

3 YR. none A3,B7 14

1

-2 MO. DR4 B14 11

12

6 YRS . none none 18 -16-

The percentage of lymphoblasts reacting with the autologous serum (LRAS) showed no correlation with the total number of lymphoblasts found in the day-5-MLC, with the number of pregnancies or with the time interval between the woman's last delivery and the date when the serum was collected. For example, the mothers in case 5 and 7, who had 3 children showed about the same percentage of LRAS (14 and 11% respectively) as the mother in case 6, who had only one child. Likewise, the percent LRAS shown by the mother in case 5, (14%), whose youngest child was 2 months old was similar to the percent LRAS displayed by the mother in case 2, (11%), whose youngest child was 32 years old. The percent LRAS was not related to the total number of lymphoblasts generated by priming the mother against the husband in a 5-day-MLC; the mother in case 1 had 45% LRAS, and the mother in case 2, 6, and 7 had 11%, 14% and 11% LRAS respec- tively, although the percent of lymphoblasts found in the 5-day-MLC with the husbands was the same (3%) in all these cases.

Binding of the serum to Al MLC-primed to an unrelated control was observed in case 1, 4 and 7. In all these cases, the "stimulation" control lymphocytes shared with the father la-like antigens (HLA-DR and/or MT) which were absent in the responder, i.e. , in the mother. Thus the autologous serum seems to react with lymphocyte receptors for the la antigens inherited by the children from their father. Maternal lymphoblasts primed against controls that shared no HLA-DR or MT antigens with the father and/or that were la compatible with the responder herself did not bind the autologous serum (case 4, 5, 8) . -17-

To determine whether MLC-primed lymphoblasts from individuals who have not been alloimmunized in vivo, also acquire the capacity of reacting with autologous serum, lymphocytes from the fathers were MLC-allo-activated against an unrelated individual, and tested for binding of autologous serum. No significant binding was observed in any of these controls.

Inhibition of anti-HLA activity by anti-idiotypic sera.

Sera from two healthy staff members who have been bled repeatedly during or following pregnancy, in a search for anti-HLA typing reagents, were used for determining whether the absence or disappearance of anti-HLA antibodies is related to anti-idiotypic antibodies.

The first case investigated is that of ES, a healthy woman from whom sera were obtained sequen¬ tially throughout her second pregnancy. The HLA genotype of ES is A2, Bw49, DRw6/A3, Bw56, DR2. The first child inherited the paternal HLA A2, Bw49 , DRw6, and maternal All, B17, DR7; the second child showed the paternal A3, Bw56, DR2 and the maternal All, B17, DR7 haplotypes. None of ES's sera reacted with T and B lymphocytes from her husband, R.S., or with lymphocytes from the ^"HLA reference panel.

The presence of anti-idiotypic antibodies, recognizing the anti-HLA-DR2 idiotype inherited by one of the children from their father, was inves- tigated by determining the capacity of ES's sera to inhibit the lymphocytotoxic activity of 2 monospeci- fic anti-HLA-DR2 alloantisera, 8W907 and COL3025. These sera displayed a titer of 1:16 when tested on purified B lymphocytes from RS and on B lymphocytes -18-

from unrelated individuals homozygous for HLA-DR2. ES's pregnancy sera inhibited to the same extent the cytotoxic activity of allo-antisera recognizing other DR specifics such as DR1, DR3, DR4, and DR5. The blocking effect was therefore specific for anti-HLA-Dr allo-antibodies. As illustrated in Fig. 1 sera collected from ES during the 5h, 7th, 8th and 9th month of pregnancy inhibited the cytotoxic acti¬ vity of the 8W907 alloantiserum by 50%. The percent inhibition exhibited by the 3rd and 6th month sera was of 80 and 100% respectively. Cytofluorometric and lymphocytotoxicity determination showed that ES^•s sera reacted with 23 to 45% of AL primed in 5-day-MLC against RS, yet failed to react with resting autologous lymphocytes (Table 2) .

OMPI -19-

Table 2. Monitoring of Anti-Idiotype Antibody in Parous Women Tested

CASE: BINDING OF SERA TO

ES AUTOLOGOUS LYMPHOBLASTS (%)

PREGNANCY SERA PRIMED AGAINST HUSBAND

3rd month 27*(29)⁺

5th month 45 (NT)

6th month 25 (34)

8th month 33 (47)

9th month 23 (17)

ARF

POST-PREGNANCY

SERA

7th month 28

9th month 20

24th month 22

48th month 8

*

Cytofluorometric analysis Indirect SRBC rosetting assay

-19A-

Table 2 (Cont.) Monitoring of Anti-Idiotype Antibody in Parous Women Tested

% INHIBITION OF THE MLR RESPONSE TO IRRADIATED: AUTOLOGOUS LYMPHOBLASTS ALLOGENEIC PBL FROM:

PRIMED AGAINST HUSBAND HUSBAND CONTROL⁵ 58 0 0 NT NT NT 66 5 0 72 0 0 NT 10 0

82 65 54

64 74 73

87 75 62

57 72 65

The Control used for allogeneic MLC shared with the immunizing donor (husband) CLASS I and CLASS II HLA antigens

-20-

The reactivity of these sera with AL was further confirmed by indirect SRBC rosetting assays. Indeed, ES's lymphoblasts which had been primed against RS and preincubated with autologous serum formed rosettes with sheep erythrocytes that have been chemically coated with purified anti-human IgG. (Table 2). This indicates that ES's sera contain autoantibodies which bind to autologous alloacti¬ vated lymphoblasts. The MLC reactivity of resting, OK.T3 positive T cells from ES against AL primed to RS or to an unrelated control, who was MT identical to RS, was significantly inhibited by ES's sera. None of these sera were, however, inhibitory in allogeneic MLC, in which resting lymphocytes from ES were stimulated with irradiated PBL from her husband or from the control (Table 2). Thus, antibodies present in ES's sera block the MLC stimulating determinants ex¬ pressed by autologous alloactivated lymphoblasts but do not bind to the immunizing donor's la antigens. The responding capacity of ES's lymphocytes to her husband was, however, inhibited by 20-40% when T lymphocytes from ES were pretreated with autologous serum and complement prior to testing. The day-5 blasts failed to react with autologous sera, indica¬ ting that the inhibition of the MLC response to the husband could be due to the deletion of specific alloreactive clones.

Since both ES and RS's mother (who has the HLA genotype A28, Bw49, DR5/A3, Bw56, DR2) were allosen- sitized to the DRw6 antigen inherited by RS from his father, the question was asked whether the idiotypic receptors for DRw6 expressed by the two women might be similar. Indeed, 59% of the lymphoblasts derived -21-

from a 5-day MLC between ES and RS, reacted with serum from RS's mother and 45% of the lymphoblasts reacted with autologous serum. Eleven percent of the lymphoblasts from RS's mother primed on a 5-day-MLC against her son reacted with autologous serum and 14% reacted with ES's serum. The anti- idiotypic antibodies which are present in these two women's sera define, therefore, a common cross- reactive set of idiotypic determinants present on anti-DR2 receptors.

The second investigated case is that of ARF, a healthy staff member, from whom sequential bleedings were obtained over a period extending from the 7th month to the 4th year following the birth of her second child. The genotype of ARF, is HLA A25, B18, DR5/A2, B18, DR7 and of her husband, GRF, is HLA Al, B7, DR2/Aw29, Bw22, DRw6. ARF, displayed strong anti-HLA-B7 and anti-Bw22 lymphocytotoxins which lasted for a period of 3 years and then disappeared. No anti-HLA-DR antibodies were found in any of ARF's sera. To determine whether the disappearance of the anti-HLA-B7 and Bw22 antibodies from ARF sera was related to the development of anti-idiotypic anti¬ bodies, the capacity of ARF's most recent serum to inhibit binding of her earliest postdelivery serum to T lymphocytes from GRF was tested by σytofluoro- metry (Figure 2) .

ARF's earliest serum reacted with T lymphocytes from GRF up to a 1:1024 dilution. The titer of this serum decreased to 1:32 when preincubated with ARF's latest serum. (Figure 2) This indicates that her anti-HLA antibodies were inhibited by anti-anti- idiotypic antibodies, prevailing in later sera. Such auto-anti-idiotypic antibodies were, however. -22-

present in all the bleedings obtained from ARF, including the ones with anti-HLA antibodies, since binding to AL primed to her husband was consistently observed (Table 2). ARF's sera inhibited both her autologous MLC to primed lymphoblasts and the allogeneic MLC with her husband, as expected in view of the coexistence of anti-HLA and anti-anti-HLA antibodies in these sera (TabTe 2) .

Equivalents Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intented to be encompassed by the following claims.

Claims

-23-CLAIMS

1. Anti-idiotypic antibody that binds to antigen receptor on the surface of human T lymphocyte.

2. Antiserum obtained from a person who has been activated against an antigen, which antisera reacts with human T lymphoblasts that have been alloactivated by said antigen.

3. Antiserum obtained from a parous mother that binds to autologous T lymphoblasts which have been alloactivated by lymphocytes of the father.

4. Antiserum obtained from a parous mother that binds autologous T lymphoblasts obtained from mixed lymphocyte culture with lymphocytes of the father.

5. Antiserum obtained from a parous mother that inhibits the mixed lymphocyte culture response exhibited by T lymphocytes against autologous T lymphoblasts which have been alloactivated in a mixed lymphocyte culture by lymphocytes of the father.

6. Anti-idiotypic antibody to alloantigen recep¬ tors on the surface of T lymphocytes that inhibit the specific reactivity of antibody produced by B lymphocytes against the allo¬ antigen which binds to said receptor.

OMPI -24-

7. Anti-idiotypic serum obtained from a parous mother that inhibits the cytotoxic activity of alloantiserum to the father's allotype inher¬ ited by the child, against B lymphocytes from the father or against B lymphocytes from unrelated persons homozygous for the allotype.

8. Anti-idiotypic serum to HLA-D2 alloantigen receptor, obtained from a parous woman, at least one of her children having inherited the HLA-D2 allotype from the father, that inhibits the ability of the anti-HLA-D2 alloantisera 8W907 and COL302 to kill B lymphocytes of the father.

9. Monoclonal anti-idiotypic antibody specific for an antigen receptor on the surface of a human T lymphocyte.

10. Monoclonal anti-idiotypic antibody of Claim 9 derived from a cloned human antibody producing cell or human hybridoma.

11. Monoclonal anti-idiotypic antibody according to Claim 9 derived from a cloned murine antibody producing cell or murine hybridoma.

12. In a method for suppressing allograft immunity, the improvement of using anti-idiotypic anti- body against antigen receptors on the surface of human T lymphocytes in order to destroy selectively the T lymphocyte responsible for graft rejection. -25-

13. In a method for suppressing allograft immunity, the improvement of treating a graft recipient with anti-idiotypic antibody against specific receptors ^"on the surface of T lymphocytes to destroy selectively those T lymphocytes posses¬ sing receptors to the foreign antigens of the allograft.

14. A method of separating human T lymphocytes having a specific idiotypic antigen receptor on their surface from a population of lymphocytes comprising using an immunoadsorbent device consisting of an anti-idiotypic antibody against the specific antigen receptor bound to a solid phase support.

15. In a method of treating autoimmune diseases, the improvement of using anti-idiotypic antibody against the antigen receptor on the surface of T lymphocytes to selectively destroy those T lymphocytes responsible for the autoimmunity.